The research proposal investigates the mechanism of protein import into the mitochondrion. The experimental model is the budding yeast Saccharomyces cerevisiae, which is an ideal model for mammalian systems because protein import is highly conserved. Previous work has identified an import pathway for proteins of the mitochondrial inner membrane, which is distinct from the pathway used by precursors with an amino-terminal targeting presequence. Components of this import pathway include the soluble Tim8p-Tim13p and Tim9p-Tim10p complexes of the intermembrane space and the TIM22 complex (Tim12p, Tim18p, Tim22p, and Tim54p) of the inner membrane. Mutations in DDP1 (deafness/dystonia protein; homologous to TimSp) cause the human disease Mohr-Tranebjaerg syndrome, which is most likely caused by a defective protein import machinery. The objective of the research proposed here is to define the molecular mechanisms of this import pathway with a combined biochemical, biophysical, and genetic approach. Specifically, the inner membrane substrates and their motifs, which are recognized by the Tim8p-Tim13p and Tim9p- Tim10p complexes, will be determined. Moreover, the mechanism by which the Tim8p-Tim13p and [unreadable] Tim9p-Tim10p complexes are assembled and subsequently escort the substrates to the inner membrane will be elucidated. In addition, assembly and maintenance of TIM22 import components will be characterized. The proposed project will expand fundamental knowledge about the mechanism of protein insertion into the mitochondrial inner membrane, extending present studies that have focused generally on how proteins reach the soluble compartments of the mitochondria. Also, these studies will contribute to the basic understanding of how proteins insert into membranes and how defects in mitochondrial biogenesis can contribute to mitochondrial diseases. [unreadable] [unreadable]